Synthesis of Poly-(L-Lactic Acid)

Synthesis of Poly-(L-Lactic Acid)

Part I - Chemical Synthesis

Materials

L-lactide monomer, (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione

8 wt% stannous octoate in hexane

Dichloromethane (CH₂Cl₂)

Magnetic stir bar: football-shaped, teflon

Liquid nitrogen

Cork stands

Material/equipment noted on diagram

Neutrad soap and acetone

Various gloves

Safety

1. When the glass reaction system is under vacuum, it is extremely vulnerable. Any crack in the glassware or any other flaw can cause a fatal destruction in the system leading to its implosion. When the system is under vacuum, CLOSE THE HOOD.

2. The manometer contains mercury. The mercury is enclosed in a chamber, but is open to the air inside the chamber. Do NOT turn the manometer upside down. Do NOT quickly change the pressure in the system as mercury can pop out of its vein in the chamber.

3. Liquid nitrogen is very cold and can cause painful burns. When handling liquid nitrogen, always wear zetex gloves (white, cold temperature-resistant gloves). Dr. Saterbak will usually handle the liquid nitrogen.

4. Hexane is a volatile, flammable solvent. Its risk codes are given as “Harmful” and “Dangerous.”

· The Chemical Toxicity and Risk Information as well as the glove recommendations for Heavy Exposure and Intermittent Exposure are included with the protocols.

· In this protocol, you will be handling very small volumes of hexane. Wear disposable nitrile gloves when handling hexane.

· Do NOT breathe the vapors. ALL work MUST be conducted in the chemical fume hood.

· Keep methanol away from ignition sources.

· If you spill hexane on your skin, wash that area immediately. Change gloves if you spill any on your gloves. Report any spills to Dr. Saterbak.

5. Dichloromethane (CH₂Cl₂) is a volatile solvent. Its risk codes are given as “Harmful” and “Dangerous.” It is also a known carcinogen in animals.

· The Chemical Toxicity and Risk Information as well as the glove recommendations for Heavy Exposure and Intermittent Exposure are included with the protocols.

· In this protocol, you will be handling moderate volumes of CH₂Cl₂. Wear Viton or NitriSolve gloves when handling CH₂Cl₂.

· Do NOT breathe the vapors. ALL work MUST be conducted in the chemical fume hood.

· If you spill CH₂Cl₂ on your skin, wash that area immediately. Rinse off any spills on the gloves, as well. Report any spills to Dr. Saterbak.

6. Acetone is a volatile, flammable solvent. Its risk codes are given as “Toxic” and “Very Dangerous.”

· The Chemical Toxicity and Risk Information as well as the glove recommendations for Heavy Exposure and Intermittent Exposure are included with the protocols.

· Keep methanol away from ignition sources.

· Since you will be using acetone to wash glassware, it will contact your gloves. Wear NitriSolve gloves when handling acetone. Do not wear Viton gloves when handling acetone.

· If you spill hexane on your skin, wash that area immediately. Rinse off any spills on the gloves, as well. Report any spills to Dr. Saterbak.

7. In Steps 1 and 16, you will be handling a hot 100 mL round bottom flask. Wear the thick orange gloves when handling the flask.

8. Glassware, such as pieces [14] and [6], are very expensive. Be very careful during assembly and washing.

9. Wear safety glasses at all times.

Experimental Set-Up

Below is a summary of the critical items of the reaction system. Refer to the attached diagram.

1 - Glass Reactor

A 100 mL round bottom flask.

4 - Glass T Connector

Connects reactor to condenser. Also allows for insertion of a thermometer to gauge the reaction vapor temperature.

5 - Thermometer

Provides the temperature of those volatile components (e.g. hexane) that are about to be condensed.

6 - Glass Condenser

Condenses volatile components of the reaction. The condenser must have cool water running through it continuously to function properly. Notice that the "Water In" line is connected to the condenser at the port furthest from the reaction. This provides for the countercurrent flow of cooling water, the most efficient design.

9 - Glass Curve Connector

Connects glass condenser to receiver flask. Also allows for a port to the vacuum line.

10 - Glass Receiver Flask

A 100 mL round bottom flask collects the volatile hexane that is condensed during the reaction. The receiver flask will collect low volumes of liquid (if any).

12 - Manometer

A manometer is a device that measures pressure. This type of manometer is read in the following manner. On the left column of mercury, read the value corresponding to the mercury height (read up from 0). On the right column of mercury, read the value corresponding to its height (read down from 0). Add the two readings. The sum is the pressure in the system.

14 - Glass Vacuum Distributor

Allows multiple items to be connected to the vacuum pump.

16 - Glass Vacuum Trap

Condenses any volatile component using the low temperature of liquid nitrogen. A trap is an absolute necessity when using a vacuum so that toxic solvents will not collect in the vacuum pump oil or be released as vapor from the vacuum pump. Notice that the line connected to the pump enters the side of the trap so that any condensed items are not pulled into the pump.

17 - Liquid Nitrogen Dewar

Contains liquid nitrogen to condense any volatile components.

18 - Vacuum Pump

The vacuum pump pulls a vacuum down to <5-10 mmHg across the system.

Instructor Preparation

1. Put liquid N₂ in carrying dewar.

2. Dissolve 0.247 g stannous octoate and 2.841 g hexane in small glass jar with lid. Use glass pipettes to transfer stannous octoate and hexane. Allow up to 30 min to dissolve. Amount prepared should be sufficient for 5-6 lab groups. Prepare fresh each week. Measurements must be done in hood because of volatility of hexane.

3. Aliquot CH₂Cl₂ to 1 L containers.

Procedure

ALL STEPS MUST BE CONDUCTED IN A CHEMICAL FUME HOOD.

1. The round bottom flask [1] and glass T connector [4] must be clean, dry and at room temperature. In general, all glassware that contacts polymer should be clean, dry and at room temperature. Neutrad soap and acetone are appropriate for cleaning glassware. The drying oven can dry the glassware quickly; however, glassware must cool before it is used. CAUTION: Glassware leaving the drying oven is hot. Use thick orange gloves when handling.

2. Assemble the experimental apparatus as shown. Confirm that all joints are well sealed with vacuum grease. Ensure that green clamps are in place, as appropriate.

3. Place 10.0 g of L-Lactide into a 100 mL glass round bottom flask [1]. L-Lactide is stored in the refrigerator. Weigh the L-Lactide on the Explorer balance in the chemical fume hood.

4. NOTE: Wear disposable nitrile gloves for this step. Add 0.475 g of the stannous octoate/hexane mixture to the 100 mL glass round bottom flask [1]. Use the Explorer balance in the chemical fume hood. Use a glass pipette to transfer the stannous octoate mixture.

5. Insert a football-shaped teflon magnetic stir bar [3] into the 100 mL glass round bottom flask [1]. Connect flask to vacuum system. Ensure all pieces and joints are in the correct place. Confirm that all joints are well sealed with vacuum grease. Ensure that green clamps are in place, as appropriate. Do NOT turn the stir bar on.

6. Confirm that water line out [8] is emptying to drain in hood. Turn on the water flow through glass condenser [6].

7. Slowly heat the reactor by adjusting the heating mantle controller (not hot plate) to level 3 (not higher).

8. Continue heating the reactor until all the components have melted (30 + 10 min). You may want to turn on the stir bar at a very slow rate for a few seconds to mix melted and solid components every 5 min. Do not be concerned if crystals condense on the side of the flask.

9. Turn on the stirrer to level 4 once the components have melted.

10. After 20-30 min of heating and stirring, begin to pull a vacuum on the system.

· Prior to any vacuum work, notify Dr. Saterbak or a T.A.

· Ensure that all valves in the glass vacuum distributor [14] are closed.

· Fill the dewar [17] surrounding the vacuum condenser [16] with liquid nitrogen.

· Turn on the vacuum pump.

· Open the valve [13] on the glass vacuum distributor [14] that leads to the manometer [12]. Observe that P<5-10 mmHg.

Ø If P>10 mmHg, there may be a leak in your system. Consult with Dr. Saterbak or a T.A. to trouble shoot.

· Once the pressure has stabilized, open any valve in the glass distributor [14] that is free/unconnected [20].

Ø Open the valve slowly.

Ø Do not let P>20 mmHg.

· Once the pressure has stabilized, open the valve [11] leading to the reaction system.

Ø Open the valve slowly. ONE PARTNER SHOULD WATCH THE REACTION VESSEL!

Ø If the reactants begin to boil violently, close the valve [11]. Consult with Dr. Saterbak or a T.A. to trouble shoot.

Ø Do not run the vacuum pump for a long time at P>20 mmHg.

· Once partial vacuum (P~20 mmHg) has been established on the reaction vessel, close the valve in the distributor [14] that is free/unconnected [20].

Ø Close the valve slowly. ONE PARTNER SHOULD WATCH THE REACTION VESSEL!

Ø If the reactants begin to boil violently, reopen the valve that is free/unconnected [20]. Consult with Dr. Saterbak or a T.A. to trouble shoot.

Ø Continue to close the valve until the system can accept full vacuum (P<5-10 mmHg).

· Pressure should remain at P<5-10 mmHg throughout experiment.

11. Let the reaction proceed under heat, stirring and vacuum. The polymer may show gentle, viscous bubbles as it reacts. Do not be concerned if the stir bar stops stirring due to the high viscosity of the material.

12. Allow the reaction to proceed for 1-1.5 hr after reduced pressure has been achieved. If the reaction mixture becomes discolored (e.g. yellow or brown), discontinue the reaction.

13. At the end of the reaction, turn off the vacuum pump.

· Prior to any vacuum work, notify Dr. Saterbak or a T.A.

· Close valve in the glass distributor [14] that leads to the reaction system [11].

· Slowly open any valve in the glass distributor [14] that is free/unconnected [20]; the pressure will quickly rise (see manometer, [12]).

· Turn off vacuum pump immediately.

· Slowly open valve in the glass distributor [14] that leads to the reaction system [11].

14. Turn off heating and stirring.

15. Turn off the water flow [8] through glass condenser [6].

16. CAUTION: The reaction system may still be under a slight vacuum. Slowly open/expose system to atmospheric air. CAUTION: The reaction vessel is hot. Use the thick orange gloves when handling. Remove round bottom flask and set on cork stand for 30 min to cool. Place a glass stopper on the flask.

17. While PLLA product is cooling, clean glass experimental assembly (pieces [4], [5], [6], [9], [10]) using Neutrad soap and acetone, as necessary. Glassware is very expensive. Please be VERY careful. Leave the dewar containing liquid nitrogen in the hood to evaporate.

18. CAUTION: Wear Viton gloves when handling CH₂Cl₂. Once PLLA is cool, add 80 mL of CH₂Cl₂ to the flask to solubilize the polymer for further purification.

· The measurement of CH₂Cl₂ and its addition to the polymer should be done in a chemical fume hood.

· As soon as the CH₂Cl₂ is added, place a glass stopper on the flask.

· To accelerate the dissolution of PLLA, the solution can be stirred. If the stir bar from the reaction is stuck in the polymer, add another. Set the flask on its cork stand on a stir plate and gently stir the mixture. Mix for 30-60 min, as necessary.

· Solubilization may take several hours.

19. While PLLA product is dissolving, finish any cleaning. Clean hood area.

20. Even if your polymer is not completely dissolved, you may leave it in a chemical fume hood (not on the stir plate) until the next session. Make sure that the round bottom flask is closed with a glass stopper. Label your flask.

Synthesis of Poly-(L-Lactic Acid)

Part II - Polymer Clean-up

Materials

Methanol

Dichloromethane (CH₂Cl₂)

Magnetic stir bar

Filter paper (No. 1)

Buchner funnel / rubber joints

Glass aspirator vessel and tubing

2 L beaker

Liquid nitrogen

Cork stands

Material/equipment noted on diagram

Neutrad soap and acetone

Various gloves

Safety

1. Methanol is a very flammable solvent. Its risk code is given as “Harmful” and “Dangerous.”

· The Chemical Toxicity and Risk Information as well as the glove recommendations for Heavy Exposure and Intermittent Exposure are included with the protocols.

· In this protocol, you will be handling large volumes of methanol. Wear Viton or NitriSolve gloves when handling methanol.

· Do NOT breathe the vapors. ALL work MUST be conducted in the chemical fume hood.

· Keep methanol away from ignition sources.

· If you spill methanol on your skin, wash that area immediately. Rinse off any spills on the gloves, as well. Report any spills to Dr. Saterbak.

2. Dichloromethane (CH₂Cl₂) is a volatile solvent. Its risk codes are given as “Harmful” and “Dangerous.” It is also a known carcinogen in animals.

· The Chemical Toxicity and Risk Information as well as the glove recommendations for Heavy Exposure and Intermittent Exposure are included with the protocols.

· In this protocol, you will be handling moderate volumes of CH₂Cl₂. Wear Viton or NitriSolve gloves when handling this chemical.

· Do NOT breathe the vapors. ALL work MUST be conducted in the chemical fume hood.

· If you spill CH₂Cl₂ on your skin, wash that area immediately. Rinse off any spills on the gloves, as well. Report any spills to Dr. Saterbak.

3. Acetone is a volatile, flammable solvent. Its risk codes are given as “Toxic” and “Very Dangerous.”

· The Chemical Toxicity and Risk Information as well as the glove recommendations for Heavy Exposure and Intermittent Exposure are included with the protocols.

· Keep methanol away from ignition sources.

· Since you will be using acetone to wash glassware, it will contact your gloves. Wear NitriSolve gloves when handling acetone.

· If you spill hexane on your skin, wash that area immediately. Rinse off any spills on the gloves, as well. Report any spills to Dr. Saterbak.

4. Wear safety glasses at all times.

Procedure

1. Confirm that PLLA is fully dissolved in CH₂Cl₂. Do not proceed until the polymer product is fully dissolved.

2. CAUTION: Wear Viton or NitriSolve gloves when handling large volumes of methanol. Pour 1 L of methanol into a 2 L beaker. Set beaker on stir plate and stir with a magnetic stir bar. Cover with parafilm until use.

3. Slowly pour PLLA/CH₂Cl₂ solution into the methanol. Pour VERY slowly; this process should take 5 min.

· PLLA should precipitate upon contact with solvent. White, stringy polymer will form - usually surrounding the stir bar.

· If the polymer does not precipitate out immediately, contact Dr. Saterbak. Sometimes, cooling the methanol on ice can improve the separation/precipitation.

4. Rinse out any remaining polymer from the sides of the glass round bottom flask using 10 mL of CH₂Cl₂. Pour the PLLA/CH₂Cl₂ solution into the methanol.

5. Allow methanol with PLLA to stir for 5-10 min. Big clumps of polymer may need to be broken up with a spatula and/or tongs.

6. While stirring, set up aspirator with Buchner funnel and Whatman filter paper. Connect the vacuum hose to the vacuum line in a chemical fume hood. Ask Dr. Saterbak or a T.A. to turn on the pump.

7. Decant as much of the methanol liquid WITHOUT polymer as possible into the jar labeled “Methanol Waste.”

8. Slowly filter the methanol/PLLA suspension using the aspirator system. Allow the PLLA to be under filtration for at least 5 min to extract as much methanol as possible.

9. Recover the solid PLLA and place in a glass beaker or jar. If needed, remove polymer from filter paper and funnel by scraping with metal spatula. You may need to discard the used filter paper and get new filter paper several times during the whole filtration process.

10. Once all the PLLA has been recovered from the methanol, dissolve the PLLA into 80 mL of CH₂Cl₂. This dissolution may take up to 15 min.

11. Repeat steps 2. – 9. Fresh methanol must be used.

12. Collect the PLLA and place in a glass beaker or jar.

13. Allow PLLA to dry in the chemical fume hood for 20 min.

14. Place PLLA into Labconco vacuum jar.

15. Attach jar to vacuum apparatus.

· Set the Labconco vacuum jar on a cork stand.

· Secure the jar to the bars at the back of the hood using clamps.

· Attach jar to vacuum apparatus (distributor and vacuum pump) by attaching the vacuum line [11] to a glass vacuum line adapter (with valve) and the adapter to the vacuum jar.

· Confirm that all joints are well sealed with vacuum grease and clamped, as necessary.

16. Using the techniques previously described, pull a P<5-10 mmHg vacuum on the PLLA in the Labconco jar for 1 hour to remove volatile solvents.

· Prior to any vacuum work, notify Dr. Saterbak or a T.A.

· Use the same set-up as before, substituting the vacuum jar for the reaction vessel.

· Turn the vacuum on and off as before.

· To release the vacuum on the PLLA sample, slowly twist the valve on the adapter to the vacuum jar.

17. Remove Labconco vacuum jar from vacuum apparatus.

18. For long-term storage of PLLA, transfer your polymer to a glass jar with lid. Store in the vacuum oven with the lid LOOSELY capped. See instructions on how to operate vacuum oven.

19. Clean the glassware (vacuum jar, adapter, [6], [9], [10], [16]). Dispose of any organic liquids properly.

20. During the next lab session, weight the polymer. Calculate the yield and/or recovery, as appropriate.